Differentiated vascular smooth muscle cells (VSMCs) undergo a unique process known as "phenotype modulation": the transition from a quiescent, "contractile" phenotype to a proliferative, "synthetic" state. This process contributes to numerous lung and vascular disorders, including lymphangioleiomyomatosis, pulmonary arterial hypertension and atherosclerosis. Our long-term goal is to elucidate the regulatory mechanisms controlling VSMC proliferation and differentiation as a prerequisite to the development of effective therapeutic protocols. The specific hypothesis is that bone morphogenetic protein (BMP) signaling shifts dedifferentiated VSMCs to a contractile phenotype, thereby contributing to the function and homeostasis of the vessel wall. We base this hypothesis on the results of in vitro studies, gene inactivation phenotypes and mutations found in human diseases, all linking BMP signaling to VSMC phenotype modulation, especially in the pulmonary vasculature. Our preliminary studies support this hypothesis and show that BMPs are potent inducers of VSMC differentiation in three different cell systems. Based on this evidence, the experimental focus of this 2-year R21 proposal is on the requirement of BMP signaling for maintenance of VSMC differentiation and its molecular mechanism of action. Specific Aim 1: Inhibit BMP signaling in cultured VSMCs via genetic inhibitors of the BMP receptors. We will correlate the expression of contractile markers with the loss of BMP influence. Specific Aim 2: Elucidate the pathways involved in BMP- mediated VSMC phenotype modulation. The p38 MAPK, RhoA and PI3K-Akt pathways can be activated by BMPs and are involved in VSMC differentiation. Using small molecule and genetic inhibitors, we will interfere with these Smad-independent pathways and with the canonical BMP-Smad 1/5/8 signal, and determine their contribution to the BMP-induced VSMC phenotype. Specific Aim 3: Characterize the mechanism of BMP-mediated regulation of VSMC-specific genes transcription. Known effectors of VSMC-specific transcription include the SRF/Myocardin/CArG-box complex and the TGF-( control element. We will examine the effects of BMP signaling on these two regulatory elements using VSMC-specific promoter-reporter constructs and small molecule or genetic inhibitors of SRF, myocardin and TGF(-specific signaling. [unreadable] [unreadable] [unreadable] [unreadable]